MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing.

Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.

Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery.

Gene expression can be activated or suppressed using CRISPR--Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome.

Pathway-Based High-Throughput Chemical Screen Identifies Compounds That Decouple Heterochromatin Transformations.

Heterochromatin protein 1 (HP1) facilitates the formation of repressive heterochromatin domains by recruiting histone lysine methyltransferase enzymes to chromatin, resulting in increased levels of histone H3K9me3. To identify chemical inhibitors of the HP1-heterochromatin gene repression pathway, we combined a cell-based assay that utilized chemical-mediated recruitment of HP1 to an endogenous active gene with high-throughput flow cytometry. Here we characterized small molecule inhibitors that block HP1-mediated heterochromatin formation. Our lead compounds demonstrated dose-dependent inhibition of HP1-stimulated gene repression and were validated in an orthogonal cell-based system. One lead inhibitor was improved by a change in stereochemistry, resulting in compound 2, which was further used to decouple the inverse relationship between H3K9 and H3K4 methylation states. We identified molecular components that bound compound 2, either directly or indirectly, by chemical affinity purification with a biotin-tagged derivative, followed by quantitative proteomic techniques. In summary, our pathway-based chemical screening approach resulted in the discovery of new inhibitors of HP1-mediated heterochromatin formation while identifying exciting new molecular interactions in the pathway to explore in the future. This modular platform can be expanded to test a wide range of chromatin modification pathways yielding inhibitors that are cell permeable and function in a physiologically relevant setting.

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Targeted Gene Repression Using Novel Bifunctional Molecules to Harness Endogenous Histone Deacetylation Activity.

Epigenome editing is a powerful method for life science research and could give rise to new therapies for diseases initiated or maintained by epigenetic dysregulation, including several types of cancers and autoimmune disorders. In addition, much is still unknown about the mechanisms by which histone-modifying proteins work in concert to properly regulate gene expression. To investigate and manipulate complex epigenetic interactions in live cells, we have developed a small molecule platform for specifically inducing gene repression and histone deacetylation at a reporter gene. We synthesized bifunctional ligands, or chemical epigenetic modifiers (CEMs), that contain two functional groups: a FK506 derivative capable of binding to a FKBP-Gal4 fusion transcription factor, and a histone deacetylase (HDAC) inhibitor that recruits HDAC-containing corepressor complexes. In our reporter cell line, which contains a GFP reporter allele upstream of a Gal4 DNA binding array in the murine Oct4 locus, our lead CEM repressed GFP expression by 50%. We also show that CEM recruitment of deacetylation activity causes marked deacetylation along our target loci. This system allowed us to detail the direct results of deacetylation to chromatin and measure the resulting gene expression in a chemically dependent and reversible manner. The CEMs system provides new insights into epigenetic gene regulation and has the potential to control disease-relevant gene regulation. The CEMs are derived from FDA-approved epigenetic modulator drugs, and use their pharmacology in a gene-specific way that avoids the toxicities and off-target effects caused by whole-cell application of these drugs.

Structure-inspired design of ß-arrestin-biased ligands for aminergic GPCRs.

Development of biased ligands targeting G protein-coupled receptors (GPCRs) is a promising approach for current drug discovery. Although structure-based drug design of biased agonists remains challenging even with an abundance of GPCR crystal structures, we present an approach for translating GPCR structural data into ß-arrestin-biased ligands for aminergic GPCRs. We identified specific amino acid-ligand contacts at transmembrane helix 5 (TM5) and extracellular loop 2 (EL2) responsible for Gi/o and ß-arrestin signaling, respectively, and targeted those residues to develop biased ligands. For these ligands, we found that bias is conserved at other aminergic GPCRs that retain similar residues at TM5 and EL2. Our approach provides a template for generating arrestin-biased ligands by modifying predicted ligand interactions that block TM5 interactions and promote EL2 interactions. This strategy may facilitate the structure-guided design of arrestin-biased ligands at other GPCRs, including polypharmacological biased ligands.

Report and Application of a Tool Compound Data Set.

Small molecule tool compounds have enabled profound advances in life science research. These chemicals are potent, cell active, and selective, and, thus, are suitable for interrogating biological processes. For these chemicals to be useful they must be correctly characterized and researchers must be aware of them. We mined the ChEMBL bioactivity database to identify high quality tool compounds in an unbiased way. We identified 407 best-in-class compounds for 278 protein targets, and these are reported in an annotated data set. Additionally, we developed informatics functions and a web application for data visualization and automated pharmacological hypothesis generation. These functions were used to predict inhibitors of the Chromobox Protein Homologue 5 (CBX5) mediated gene repression pathway that currently lacks appropriate inhibitors. The predictions were subsequently validated by a highly specific cell based assay, revealing new chemical modulators of CBX5-mediated heterochromatin formation. This data set and associated functions will help researchers make the best use of these valuable compounds.

Development of a S-adenosylmethionine analog that intrudes the RNA-cap binding site of Zika methyltransferase.

The Zika virus (ZIKV) has emerged as a major health hazard. We present here a high resolution structure (1.55 Å) of ZIKV NS5 methyltransferase bound to a novel S-adenosylmethionine (SAM) analog in which a 4-fluorophenyl moiety substitutes for the methyl group. We show that the 4-fluorophenyl moiety extends into a portion of the RNA binding tunnel that typically contains the adenosine 2'OH of the RNA-cap moiety. Together, the new SAM analog and the high-resolution crystal structure are a step towards the development of antivirals against ZIKV and other flaviviruses.

Structure-Based Design of a Covalent Inhibitor of the SET Domain-Containing Protein 8 (SETD8) Lysine Methyltransferase.

Selective inhibitors of protein lysine methyltransferases, including SET domain-containing protein 8 (SETD8), are highly desired, as only a fraction of these enzymes are associated with high-quality inhibitors. From our previously discovered SETD8 inhibitor, we developed a more potent analog and solved a cocrystal structure, which is the first crystal structure of SETD8 in complex with a small-molecule inhibitor. This cocrystal structure allowed the design of a covalent inhibitor of SETD8 (MS453), which specifically modifies a cysteine residue near the inhibitor binding site, has an IC50 value of 804 nM, reacts with SETD8 with near-quantitative yield, and is selective for SETD8 against 28 other methyltransferases. We also solved the crystal structure of the covalent inhibitor in complex with SETD8. This work provides atomic-level perspective on the inhibition of SETD8 by small molecules and will help identify high-quality chemical probes of SETD8.

Discovery of G Protein-Biased D2 Dopamine Receptor Partial Agonists.

Biased ligands (also known as functionally selective ligands) of G protein-coupled receptors are valuable tools for dissecting the roles of G protein-dependent and independent signaling pathways in health and disease. Biased ligands have also been increasingly pursued by the biomedical community as promising therapeutics with improved efficacy and reduced side effects compared with unbiased ligands. We previously discovered first-in-class ß-arrestin-biased agonists of dopamine D2 receptor (D2R) by extensively exploring multiple regions of aripiprazole, a balanced D2R agonist. In our continuing efforts to identify biased agonists of D2R, we unexpectedly discovered a G protein-biased agonist of D2R, compound 1, which is the first G protein-biased D2R agonist from the aripiprazole scaffold. We designed and synthesized novel analogues to explore two regions of 1 and conducted structure-functional selectivity relationship (SFSR) studies. Here we report the discovery of 1, findings from our SFSR studies, and characterization of novel G protein-biased D2R agonists.

Non-Substrate Based, Small Molecule Inhibitors of the Human Isoprenylcysteine Carboxyl Methyltransferase.

Activating mutations of human K-Ras proteins are among the most common oncogenic mutations, present in approximately 30% of all human cancers. Posttranslational modifications to K-Ras guide it to the plasma membrane and disruption of this localization inhibits the growth of Ras-driven cancers. The human isoprenylcysteine carboxyl methyltransferase (hIcmt) enzyme catalyzes the final α-carboxyl methylesterification of the C-terminal farnesyl cysteine of K-Ras, which is necessary for its proper localization. Thus, hIcmt inhibition is a regarded as a promising cancer therapy. A high quality inhibitor of hIcmt with in vivo activity would advance hIcmt research and drug development. Herein, Wwe report the results of a screen for small molecule hIcmt inhibitors in a library of molecules that were not hIcmt substrate analogs. The lead compound identified by this screen (1) was modified to remove chemical liabilities and to increase potency. The most potent resulting compound (5) inhibited hIcmt in vitro with low micromolar potency (IC50 = 1.5 ± 0.2 µM) and was kinetically characterized as a competitive inhibitor for prenylated substrates and a non-competitive inhibitor for the cofactor and methyl donor S-adenosylmethionine (SAM). These inhibitors offer important structure activity relationships for the future development of hIcmt inhibitors with in vivo activity.

A Potent, Selective, and Cell-Active Inhibitor of Human Type I Protein Arginine Methyltransferases.

Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and the pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective, and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical, and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, -3, -4, -6, and -8 but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease.

Discovery of a selective, substrate-competitive inhibitor of the lysine methyltransferase SETD8.

The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure-activity relationships (SAR) of this series.

Structure-activity relationship studies of SETD8 inhibitors.

SETD8 (also known as SET8, PR-SET7, or KMT5A (lysine methyltransferase 5A)) is the only known lysine methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). In addition to H4K20, SETD8 monomethylates non-histone substrates such as the tumor suppressor p53 and proliferating cell nuclear antigen (PCNA). Because of its role in regulating diverse biological processes, SETD8 has been pursued as a potential therapeutic target. We recently reported the first substrate-competitive SETD8 inhibitor, UNC0379 (1), which is selective for SETD8 over 15 other methyltransferases. We characterized this inhibitor in a battery of biochemical and biophysical assays. Here we describe our comprehensive structure-activity relationship (SAR) studies of this chemical series. In addition to 2- and 4-substituents, we extensively explored 6- and 7-substituents of the quinazoline scaffold. These SAR studies led to the discovery of several new compounds, which displayed similar potencies as compound 1, and interesting SAR trends.

Second-generation histone deacetylase 6 inhibitors enhance the immunosuppressive effects of Foxp3+ T-regulatory cells.

Second-generation Tubastatin A analogues were synthesized and evaluated for their ability to inhibit selectively histone deacetylase 6 (HDAC6). Substitutions to the carboline cap group were well-tolerated with substitution at the 2-position of both ß- and γ-carbolines being optimal for HDAC6 activity and selectivity. Some compounds in this series were determined to have subnanomolar activity at HDAC6 with more than 7000 fold selectivity for HDAC6 versus HDAC1. Selected compounds were then evaluated for their ability to augment the immunosuppressive effect of Foxp3+ regulatory T cells. All compounds tested were found to enhance the ability of regulatory T cells to inhibit the mitotic division of effector T cells both in vitro and in vivo, suggesting that further investigation into the use of these compounds for the treatment of autoimmune disorders is warranted.

Rational design and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin A.

Structure-based drug design combined with homology modeling techniques were used to develop potent inhibitors of HDAC6 that display superior selectivity for the HDAC6 isozyme compared to other inhibitors. These inhibitors can be assembled in a few synthetic steps, and thus are readily scaled up for in vivo studies. An optimized compound from this series, designated Tubastatin A, was tested in primary cortical neuron cultures in which it was found to induce elevated levels of acetylated alpha-tubulin, but not histone, consistent with its HDAC6 selectivity. Tubastatin A also conferred dose-dependent protection in primary cortical neuron cultures against glutathione depletion-induced oxidative stress. Importantly, when given alone at all concentrations tested, this hydroxamate-containing HDAC6-selective compound displayed no neuronal toxicity, thus, forecasting the potential application of this agent and its analogues to neurodegenerative conditions.

Stereoselective HDAC inhibition from cysteine-derived zinc-binding groups.

A series of small-molecule histone deacetylase (HDAC) inhibitors, which feature zinc binding groups derived from cysteine, were synthesized. These inhibitors were tested against multiple HDAC isoforms, and the most potent, compound 10, was determined to have IC(50) values below 1 microM. The compounds were also tested in a cellular assay of oxidative stress-induced neurodegeneration. Many of the inhibitors gave near-complete protection against cell death at 10 microM without the neurotoxicity seen with hydroxamic acid-based inhibitors, and were far more neuroprotective than HDAC inhibitors currently in clinical trials. Both enantiomers of cysteine were used in the synthesis of a variety of novel zinc-binding groups (ZBGs). Derivatives of L-cysteine were active in the HDAC inhibition assays, while the derivatives of D-cysteine were inactive. Notably, the finding that both the D- and L-cysteine derivatives were active in the neuroprotection assays suggests that multiple mechanisms are working to protect the neurons from cell death. Molecular modeling was employed to investigate the differences in inhibitory activity between the HDAC inhibitors generated from the two enantiomeric forms of cysteine.

Novel inhibitors of human histone deacetylase (HDAC) identified by QSAR modeling of known inhibitors, virtual screening, and experimental validation.

Inhibitors of histone deacetylases (HDACIs) have emerged as a new class of drugs for the treatment of human cancers and other diseases because of their effects on cell growth, differentiation, and apoptosis. In this study we have developed several quantitative structure-activity relationship (QSAR) models for 59 chemically diverse histone deacetylase class 1 (HDAC1) inhibitors. The variable selection k nearest neighbor (kNN) and support vector machines (SVM) QSAR modeling approaches using both MolconnZ and MOE chemical descriptors generated from two-dimensional rendering of compounds as chemical graphs have been employed. We have relied on a rigorous model development workflow including the division of the data set into training, test, and external sets and extensive internal and external validation. Highly predictive QSAR models were generated with leave-one-out cross-validated (LOO-CV) q2 and external R2 values as high as 0.80 and 0.87, respectively, using the kNN/MolconnZ approach and 0.93 and 0.87, respectively, using the SVM/MolconnZ approach. All validated QSAR models were employed concurrently for virtual screening (VS) of an in-house compound collection including 9.5 million molecules compiled from the ZINC7.0 database, the World Drug Index (WDI) database, the ASINEX Synergy libraries, and other commercial databases. VS resulted in 45 structurally unique consensus hits that were considered novel putative HDAC1 inhibitors. These computational hits had several novel structural features that were not present in the original data set. Four computational hits with novel scaffolds were tested experimentally, and three of them were confirmed active against HDAC1, with IC50 values for the most active compound of 1.00 microM. The fourth compound was later identified to be a selective inhibitor of HDAC6, a Class II HDAC. Moreover, two of the confirmed hits are marketed drugs, which could potentially facilitate their further development as anticancer agents. This study illustrates the power of the combined QSAR-VS method as a general approach for the effective identification of structurally novel bioactive compounds.

Chemical origins of isoform selectivity in histone deacetylase inhibitors.

Histones undergo extensive posttranslational modifications that affect gene expression. Acetylation is a key histone modification that is primarily regulated by two enzymes, one of which is histone deacetylase (HDAC). The activity of HDAC causes transcriptional silencing of DNA. Eleven distinct zinc-dependent histone deacetylase isoforms have been identified in humans. Each isoform has a unique structure and function, and regulates a unique set of genes. HDAC is responsible for the regulation of many genes involved in cancer cell proliferation, and it has been implicated in the pathogenesis of many neurological conditions. HDAC inhibitors are known to be very effective anti-cancer agents, and research has shown them to be potential treatments for many other conditions. Histone deacetylase inhibitors modify the expression of many genes, and it is possible that inhibition of one isoform could cause epigenetic changes that are beneficial to treatment of a disease, while inhibition of another isoform could cause contradictory changes. Selective HDAC inhibitors will be better able to avoid these types of situations than non-specific inhibitors, and may also be less toxic than pan-HDAC inhibitors. Many potent pan-HDAC inhibitors have already been developed, leaving the development of selective inhibitors at the forefront of HDAC drug development. Certain structural moieties may be added to HDAC inhibitors to give isoform selectivity, and these will be discussed in this review. This review will focus on the applications of selective HDAC inhibitors, inhibitors reported to show selectivity, and the relationship between inhibitor structure and selectivity.